Purpose:
Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) play a key role in various non-image forming functions, such as circadian photoentrainment and acute masking of locomotor activity by light. In addition, ipRGCs mediate retrograde visual signaling in the retina. We recently demonstrated in the mouse that ipRGCs express all of the core components of the mammalian circadian clock. Here we tested the functionality of the ipRGC clock in an ipRGC-specific circadian-clock-deficient mouse model.

Methods:
An ipRGC-specific circadian-clock-deficient mouse line was generated by conditionally knocking out the essential circadian clock component Bmal1 in ipRGCs using the Cre-loxP system. Specifically, we crossed Bmal1f/f mice with Opn4Cre/Cre;Z/G mice. Immunohistochemistry was used to label cell markers and assay the expression of BMAL1 in retinal cells. Light entrainment and negative masking of the voluntary locomotor activity rhythm was tested using activity monitoring with wheeled cages. We measured spatial frequency threshold (i.e. acuity) and contrast sensitivity of freely moving mice by observing their optomotor responses to moving sine-wave gratings, using the Optomotry system.

Results:
In the Bmal1f/f;Opn4Cre/+;Z/G retina, BMAL1 expression pattern was normal among retinal cells but below detection threshold in ipRGCs. We did not find any obvious morphological defects in the retinal tissue in this line, at least at 2 months of age. However, we were unable to detect the somatas of ipRGCs in Bmal1f/f;Opn4Cre/+;Z/G retinas using an antibody against melanopsin. Yet, ipRGC cells showed normal levels of GFP expression, indicating that ipRGCs are present and melanopsin expression is considerably reduced in these animals. In addition, the locomotor activity rhythm of Bmal1f/f;Opn4Cre/+;Z/G animals was normally entrained by light but showed abnormal increased activity during the light phase, thus suggesting a decrease in the negative masking effect of light on locomotor behavior. Finally, Bmal1f/f;Opn4Cre/+;Z/G mice showed an altered circadian rhythm in contrast sensitivity, with a decrease during the day. Acuity was normal in Bmal1f/f;Opn4Cre/+;Z/G mice.

Conclusions:
Our data provide evidence that a functional circadian clock in ipRGCs is required for normal function of both the image-forming and the non-image-forming visual systems.